A single institution anesthetic experience with catheterization of pediatric pulmonary hypertension patients.

Cardiac catheterization remains the gold standard for the diagnosis and management of pediatric pulmonary hypertension (PH). There is lack of consensus regarding optimal anesthetic and airway regimen. This retrospective study describes the anesthetic/airway experience of our single center cohort of pediatric PH patients undergoing catheterization, in which obtaining hemodynamic data during spontaneous breathing is preferential. A total of 448 catheterizations were performed in 232 patients. Of the 379 cases that began with a natural airway, 274 (72%) completed the procedure without an invasive airway, 90 (24%) received a planned invasive airway, and 15 (4%) required an unplanned invasive airway. Median age was 3.4 years (interquartile range [IQR] 0.7-9.7); the majority were either Nice Classification Group 1 (48%) or Group 3 (42%). Vasoactive medications and cardiopulmonary resuscitation were required in 14 (3.7%) and eight (2.1%) cases, respectively; there was one death. Characteristics associated with use of an invasive airway included age <1 year, Group 3, congenital heart disease, trisomy 21, prematurity, bronchopulmonary dysplasia, WHO functional class III/IV, no PH therapy at time of case, preoperative respiratory support, and having had an intervention (p < 0.05). A composite predictor of age <1 year, Group 3, prematurity, and any preoperative respiratory support was significantly associated with unplanned airway escalation (26.7% vs. 6.9%, odds ratio: 4.9, confidence interval: 1.4-17.0). This approach appears safe, with serious adverse event rates similar to previous reports despite the predominant use of natural airways. However, research is needed to further investigate the optimal anesthetic regimen and respiratory support for pediatric PH patients undergoing cardiac catheterization.

A disrupted compartment boundary underlies abnormal cardiac patterning and congenital heart defects.

Failure of septation of the interventricular septum (IVS) is the most common congenital heart defect (CHD), but mechanisms for patterning the IVS are largely unknown. We show that a Tbx5+/Mef2cAHF+ progenitor lineage forms a compartment boundary bisecting the IVS. This coordinated population originates at a first- and second heart field interface, subsequently forming a morphogenetic nexus. Ablation of Tbx5+/Mef2cAHF+ progenitors cause IVS disorganization, right ventricular hypoplasia and mixing of IVS lineages. Reduced dosage of the CHD transcription factor TBX5 disrupts boundary position and integrity, resulting in ventricular septation defects (VSDs) and patterning defects, including Slit2 and Ntn1 misexpression. Reducing NTN1 dosage partly rescues cardiac defects in Tbx5 mutant embryos. Loss of Slit2 or Ntn1 causes VSDs and perturbed septal lineage distributions. Thus, we identify essential cues that direct progenitors to pattern a compartment boundary for proper cardiac septation, revealing new mechanisms for cardiac birth defects.

Cell Layers: uncovering clustering structure in unsupervised single-cell transcriptomic analysis.

Motivation: Unsupervised clustering of single-cell transcriptomics is a powerful method for identifying cell populations. Static visualization techniques for single-cell clustering only display results for a single resolution parameter. Analysts will often evaluate more than one resolution parameter but then only report one. Results: We developed Cell Layers, an interactive Sankey tool for the quantitative investigation of gene expression, co-expression, biological processes and cluster integrity across clustering resolutions. Cell Layers enhances the interpretability of single-cell clustering by linking molecular data and cluster evaluation metrics, providing novel insight into cell populations. Availability and implementation: https://github.com/apblair/CellLayers.

Brahma safeguards canalization of cardiac mesoderm differentiation.

Differentiation proceeds along a continuum of increasingly fate-restricted intermediates, referred to as canalization1,2. Canalization is essential for stabilizing cell fate, but the mechanisms that underlie robust canalization are unclear. Here we show that the BRG1/BRM-associated factor (BAF) chromatin-remodelling complex ATPase gene Brm safeguards cell identity during directed cardiogenesis of mouse embryonic stem cells. Despite the establishment of a well-differentiated precardiac mesoderm, Brm-/- cells predominantly became neural precursors, violating germ layer assignment. Trajectory inference showed a sudden acquisition of a non-mesodermal identity in Brm-/- cells. Mechanistically, the loss of Brm prevented de novo accessibility of primed cardiac enhancers while increasing the expression of neurogenic factor POU3F1, preventing the binding of the neural suppressor REST and shifting the composition of BRG1 complexes. The identity switch caused by the Brm mutation was overcome by increasing BMP4 levels during mesoderm induction. Mathematical modelling supports these observations and demonstrates that Brm deletion affects cell fate trajectory by modifying saddle-node bifurcations2. In the mouse embryo, Brm deletion exacerbated mesoderm-deleted Brg1-mutant phenotypes, severely compromising cardiogenesis, and reveals an in vivo role for Brm. Our results show that Brm is a compensable safeguard of the fidelity of mesoderm chromatin states, and support a model in which developmental canalization is not a rigid irreversible path, but a highly plastic trajectory.

Interactive anesthesiology educational program improves wellness for anesthesiologists and their children.

STUDY OBJECTIVE: Anesthesiologists have a high prevalence of burnout with adverse effects on professionalism and safety. The objective of this study was to assess the impact of an interactive anesthesiology educational program on the wellness of anesthesia providers and their children, as assessed by a modified Professional Fulfillment Index. DESIGN: Prospective observational study. SETTING: Perioperative area. PATIENTS: Thirty clinicians participated in the program. Twenty respondents, representing 67% of participants and each corresponding to a parent and their child or children, completed the post-event survey. INTERVENTIONS: An interactive anesthesiology educational program incorporating children, between the ages of five and eighteen years old, of anesthesia providers was held in the perioperative area. The program was held over four hours and was comprised of four sessions including pediatric anesthesia, neuroanesthesia, airway, and ultrasound stations. MEASUREMENTS: Anesthesia providers and their children were administered a post-event assessment, including a modified Professional Fulfillment Index and satisfaction survey. MAIN RESULTS: All twenty (100%) of respondents indicated it was "very true" or "completely true" that their child was happy with the program, and that it was worthwhile and satisfying to both the anesthesia provider and their child. Nineteen (95%) of reporting participants indicated it was "very true" or "completely true" that it was meaningful to have the department host such a program and 17 (85%) respondents felt their child now better understands the anesthesia work of the parent. All clinician volunteers indicated it was "very true" or "completely true" that they were contributing professionally during the program in ways that they valued most. CONCLUSION: An interactive educational wellness initiative provides an effective and feasible method for increasing professional fulfillment and satisfaction among anesthesia providers while educating our youngest generation of learners. Implementation of such a program may also occur with modifications such as televideo to maintain COVID-19 precautions.

Modeling Human TBX5 Haploinsufficiency Predicts Regulatory Networks for Congenital Heart Disease.

Haploinsufficiency of transcriptional regulators causes human congenital heart disease (CHD); however, the underlying CHD gene regulatory network (GRN) imbalances are unknown. Here, we define transcriptional consequences of reduced dosage of the CHD transcription factor, TBX5, in individual cells during cardiomyocyte differentiation from human induced pluripotent stem cells (iPSCs). We discovered highly sensitive dysregulation of TBX5-dependent pathways-including lineage decisions and genes associated with heart development, cardiomyocyte function, and CHD genetics-in discrete subpopulations of cardiomyocytes. Spatial transcriptomic mapping revealed chamber-restricted expression for many TBX5-sensitive transcripts. GRN analysis indicated that cardiac network stability, including vulnerable CHD-linked nodes, is sensitive to TBX5 dosage. A GRN-predicted genetic interaction between Tbx5 and Mef2c, manifesting as ventricular septation defects, was validated in mice. These results demonstrate exquisite and diverse sensitivity to TBX5 dosage in heterogeneous subsets of iPSC-derived cardiomyocytes and predicts candidate GRNs for human CHDs, with implications for quantitative transcriptional regulation in disease.

Minimal in vivo requirements for developmentally regulated cardiac long intergenic non-coding RNAs.

Long intergenic non-coding RNAs (lincRNAs) have been implicated in gene regulation, but their requirement for development needs empirical interrogation. We computationally identified nine murine lincRNAs that have developmentally regulated transcriptional and epigenomic profiles specific to early heart differentiation. Six of the nine lincRNAs had in vivo expression patterns supporting a potential function in heart development, including a transcript downstream of the cardiac transcription factor Hand2, which we named Handlr (Hand2-associated lincRNA), Rubie and Atcayos We genetically ablated these six lincRNAs in mouse, which suggested genomic regulatory roles for four of the cohort. However, none of the lincRNA deletions led to severe cardiac phenotypes. Thus, we stressed the hearts of adult Handlr and Atcayos mutant mice by transverse aortic banding and found that absence of these lincRNAs did not affect cardiac hypertrophy or left ventricular function post-stress. Our results support roles for lincRNA transcripts and/or transcription in the regulation of topologically associated genes. However, the individual importance of developmentally specific lincRNAs is yet to be established. Their status as either gene-like entities or epigenetic components of the nucleus should be further considered.

The E3 ubiquitin ligase Nedd4/Nedd4L is directly regulated by microRNA 1.

miR-1 is a small noncoding RNA molecule that modulates gene expression in heart and skeletal muscle. Loss of Drosophila miR-1 produces defects in somatic muscle and embryonic heart development, which have been partly attributed to miR-1 directly targeting Delta to decrease Notch signaling. Here, we show that overexpression of miR-1 in the fly wing can paradoxically increase Notch activity independently of its effects on Delta. Analyses of potential miR-1 targets revealed that miR-1 directly regulates the 3'UTR of the E3 ubiquitin ligase Nedd4 Analysis of embryonic and adult fly heart revealed that the Nedd4 protein regulates heart development in Drosophila Larval fly hearts overexpressing miR-1 have profound defects in actin filament organization that are partially rescued by concurrent overexpression of Nedd4. These results indicate that miR-1 and Nedd4 act together in the formation and actin-dependent patterning of the fly heart. Importantly, we have found that the biochemical and genetic relationship between miR-1 and the mammalian ortholog Nedd4-like (Nedd4l) is evolutionarily conserved in the mammalian heart, potentially indicating a role for Nedd4L in mammalian postnatal maturation. Thus, miR-1-mediated regulation of Nedd4/Nedd4L expression may serve to broadly modulate the trafficking or degradation of Nedd4/Nedd4L substrates in the heart.

Single-Cell Resolution of Temporal Gene Expression during Heart Development.

Activation of complex molecular programs in specific cell lineages governs mammalian heart development, from a primordial linear tube to a four-chamber organ. To characterize lineage-specific, spatiotemporal developmental programs, we performed single-cell RNA sequencing of >1,200 murine cells isolated at seven time points spanning embryonic day 9.5 (primordial heart tube) to postnatal day 21 (mature heart). Using unbiased transcriptional data, we classified cardiomyocytes, endothelial cells, and fibroblast-enriched cells, thus identifying markers for temporal and chamber-specific developmental programs. By harnessing these datasets, we defined developmental ages of human and mouse pluripotent stem-cell-derived cardiomyocytes and characterized lineage-specific maturation defects in hearts of mice with heterozygous mutations in Nkx2.5 that cause human heart malformations. This spatiotemporal transcriptome analysis of heart development reveals lineage-specific gene programs underlying normal cardiac development and congenital heart disease.

Investigating the transcriptional control of cardiovascular development.

Transcriptional regulation of thousands of genes instructs complex morphogenetic and molecular events for heart development. Cardiac transcription factors choreograph gene expression at each stage of differentiation by interacting with cofactors, including chromatin-modifying enzymes, and by binding to a constellation of regulatory DNA elements. Here, we present salient examples relevant to cardiovascular development and heart disease, and review techniques that can sharpen our understanding of cardiovascular biology. We discuss the interplay between cardiac transcription factors, cis-regulatory elements, and chromatin as dynamic regulatory networks, to orchestrate sequential deployment of the cardiac gene expression program.

Screening and biochemical analysis of GATA4 sequence variations identified in patients with congenital heart disease.

Few known monogenic causes of non-syndromic congenital heart disease (CHD) have been identified. Mutations in NKX2.5 were initially implicated in familial cases of cardiac septal defects and subsequently, functionally significant NKX2.5 mutations were found in diverse forms of non-syndromic CHD. Similarly, mutations in GATA4, which encodes a cardiac transcription factor, were first identified in familial cases of cardiac septal defects. We hypothesize that individuals with non-syndromic CHD may harbor GATA4 mutations and that these mutations alter the biochemical properties of the protein. The coding region encompassing the six exons of GATA4 was screened in a study population of 157 patients with CHD. We identified several sequence variations in GATA4. We tested these novel sequence variations that altered evolutionarily conserved amino acids and other previously reported GATA4 mutations in various biochemical assays. The novel sequence variations had no biochemical deficits while a previously reported, but unstudied, missense mutation in GATA4 (S52F) functioned as a hypomorph in transactivation assays. We did not identify any novel GATA4 mutations in our patient population with non-syndromic CHD. Consistent with previous findings, GATA4 mutations that result in deficits in transactivation ability are consistently associated with CHD suggesting that normal transactivation properties of GATA4 are required for proper cardiac development.

Hrt and Hes negatively regulate Notch signaling through interactions with RBP-Jkappa.

Notch signaling is central to cell differentiation, organ development, and apoptosis. Upon ligand binding, the Notch intracellular domain (NotchIC) translocates to the nucleus to interact with its DNA-binding partner, RBP-Jkappa. The NotchIC-RBP-Jkappa complex activates target genes, such as those encoding the Hrt and Hes families of basic-helix-loop-helix (bHLH) transcriptional repressors. Hrt transcripts are enriched in the developing cardiovascular system, and mice lacking Hrt2 have cardiac malformations. Here we show that Hrt2 and Hes1 interact with RBP-Jkappa to negatively regulate Notch-dependent activation of Hrt and Hes expression. The bHLH domain of Hrt2 was necessary for this interaction, and disrupting the protein complex abrogated the negative autoregulation. The interaction did not interfere with the formation or DNA-binding of the NotchIC-RBP-Jkappa complex, indicating direct inhibition by Hrt and Hes as co-repressors. These findings suggest a novel mechanism for negative feedback on Notch signaling that requires RBP-Jkappa to interact physically with Hrt and Hes.

Cooperative and antagonistic interactions between Sall4 and Tbx5 pattern the mouse limb and heart.

Human mutations in TBX5, a gene encoding a T-box transcription factor, and SALL4, a gene encoding a zinc-finger transcription factor, cause similar upper limb and heart defects. Here we show that Tbx5 regulates Sall4 expression in the developing mouse forelimb and heart; mice heterozygous for a gene trap allele of Sall4 show limb and heart defects that model human disease. Tbx5 and Sall4 interact both positively and negatively to finely regulate patterning and morphogenesis of the anterior forelimb and heart. Thus, a positive and negative feed-forward circuit between Tbx5 and Sall4 ensures precise patterning of embryonic limb and heart and provides a unifying mechanism for heart/hand syndromes.

Hairy-related transcription factors inhibit GATA-dependent cardiac gene expression through a signal-responsive mechanism.

Combinatorial actions of transcription factors in multiprotein complexes dictate gene expression profiles in cardiac development and disease. The Hairy-related transcription factor (HRT) family of basic helix-loop-helix proteins is composed of transcriptional repressors highly expressed in the cardiovascular system. However, it has remained unclear whether HRT proteins modulate gene expression driven by cardiac transcriptional activators. Here, we have shown that HRT proteins inhibit cardiac gene transcription by interfering with GATA transcription factors that are implicated in cardiac development and hypertrophy. HRT proteins inhibited GATA-dependent transcriptional activation of cardiac gene promoters such as the atrial natriuretic factor (ANF) promoter. Adenovirus-mediated expression of Hrt2 suppressed mRNA expression of ANF and other cardiac-specific genes in cultured cardiomyocytes. Among various signaling molecules implicated in cardiomyocyte growth, constitutively active Akt1/protein kinase B alpha relieved Hrt2-mediated inhibition of GATA-dependent transcription. HRT proteins physically interacted with GATA proteins, and the basic domain of HRT was critical for physical association as well as transcriptional inhibition. These results suggest that HRT proteins may regulate specific sets of cardiac genes by modulating the function of GATA proteins and other cardiac transcriptional activators in a signal-dependent manner.

GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5.

Congenital heart defects (CHDs) are the most common developmental anomaly and are the leading non-infectious cause of mortality in newborns. Only one causative gene, NKX2-5, has been identified through genetic linkage analysis of pedigrees with non-syndromic CHDs. Here, we show that isolated cardiac septal defects in a large pedigree were linked to chromosome 8p22-23. A heterozygous G296S missense mutation of GATA4, a transcription factor essential for heart formation, was found in all available affected family members but not in any control individuals. This mutation resulted in diminished DNA-binding affinity and transcriptional activity of Gata4. Furthermore, the Gata4 mutation abrogated a physical interaction between Gata4 and TBX5, a T-box protein responsible for a subset of syndromic cardiac septal defects. Conversely, interaction of Gata4 and TBX5 was disrupted by specific human TBX5 missense mutations that cause similar cardiac septal defects. In a second family, we identified a frame-shift mutation of GATA4 (E359del) that was transcriptionally inactive and segregated with cardiac septal defects. These results implicate GATA4 as a genetic cause of human cardiac septal defects, perhaps through its interaction with TBX5.